固定化及透性化对瑞士乳杆菌L7生产共轭亚油酸的影响

发布时间：2018-10-10 13:55

【摘要】：共轭亚油酸(conjugated linoleic acid, CLA)是一系列含共轭双键的亚油酸的位置(8,10-、9,11-、10,12-、11,13-)和几何(cis, cis; cis, trans; trans, cis; trans, trans)异构体的混合物的总称。CLA具有抗癌、抗肿瘤、提高免疫力、减少脂肪沉积等生理功能。在CLA的多种异构体中,9c,11t-CLA和10t,12c-CLA均具有独特的生理活性。天然CLA主要存在于反刍动物的肉和乳制品中且含量很少,因此,利用生物转化法制备CLA是一个很好的途径。 以本实验室分离保存的一株高产9c,11t-CLA的瑞士乳杆菌L7(L. helveticusL7)为材料,首先通过发酵条件优化,提高亚油酸(LA)在发酵液中的生物转化率,增加9c,11t-CLA的产量,进而对其细胞进行透性化处理以提高细胞膜的通透性,并对透性化处理细胞进行固定化,研究了固定化细胞的理化性质和转化特性。主要结果如下： 1)优化了菌种发酵产9c,11t-CLA的最佳条件。确定L. helveticus L7发酵的最适菌体浓度为5×1010CFU/mL左右。在发酵进行到36h时发酵液中的9c,11t-CLA达到最高值。底物LA的最适添加浓度为1mg/mL。 2)对L. helveticus L7细胞透性化条件进行了研究。分别使用十六烷基三甲基溴化铵(CTAB)、乙醚、乙醇、CTAB、甲苯以及溶菌酶对L. helveticus L7进行透性化处理,结果表明：透性化处理的效果最好的是20%的乙醇,室温下处理30min,制得的透性化细胞的产率达到468μg/mL;对超声波细胞破碎法制得的亚油酸异构酶粗酶液的操作稳定性和贮藏稳定性进行研究,结果表明：粗酶液的酶活操作稳定性较差；保存于4℃时酶活性逐步降低,120h后酶活降至初始活性的35%左右。经溶菌酶处理的透性化细胞转化率也较高。透性化细胞的酶活稳定性较粗酶液有较大提高。 3)应用响应面方法对透性化细胞的最佳固定化的条件进行了优化。结果表明海藻酸钙胶珠最佳固定化条件为：海藻酸钠浓度1.78%、氯化钙浓度3.13%、固定化时间60.30min,利用在此条件下制备所得的固定化胶珠进行发酵,9c,11t-CLA的产量达到561μg/mL。与未固定细胞相比,固定化细胞单次9c,11t-CLA产量没有明显的变化,但固定化细胞可多次重复利用,重复利用5次后活力没有明显降低,9c,11t-CLA产量保持在549μg/mL。于4℃贮存120h后酶活无明显下降,保留在90%以上,具有较好的贮藏稳定性。电镜观察发现,固定化胶珠表面及内部为错综复杂的网状结构,能够很好的满足底物和产物的传递。实验结果表明：经海藻酸钙固定,可以有效提高菌体的重复利用率,从而降低9c,11t-CLA的生产成本。
[Abstract]:Conjugated linoleic acid (conjugated linoleic acid, CLA) is a mixture of a series of linoleic acids containing conjugated double bonds (810 ~ (-10) ~ (9) C ~ (11) ~ (10) ~ (10) ~ (10) and geometric (cis, cis; cis, trans; trans, cis; trans, trans) isomers. CLA has the physiological functions of anti-cancer, anti-tumor, improving immunity, reducing fat deposition and so on. Among the various isomers of CLA, both 9ct 11t-CLA and 10tl 12c-CLA have unique physiological activities. Natural CLA mainly exists in ruminant meat and dairy products and the content is very small. Therefore, bioconversion is a good way to prepare CLA. A strain of Lactobacillus helittidis L7 (L7) with high yield of 9cnc-11t-CLA was isolated and preserved in our laboratory. The fermentation conditions were optimized to improve the bioconversion of linoleic acid (LA) in the fermentation broth and to increase the yield of 9cnc-11t-CLA. In order to improve the permeability of the cell membrane and immobilize the cells, the physicochemical properties and transformation characteristics of the immobilized cells were studied. The main results are as follows: 1) the optimum fermentation conditions of 9 cu 11 t CLA were optimized. The optimum concentration of L. helveticus L7 was about 5 脳 1010CFU/mL. When fermentation lasted for 36 h, the maximum value of 9cnc-11t-CLA in the fermentation broth was reached. The optimal concentration of substrate LA was 1 mg / mL.2) the condition of L. helveticus L7 cell permeability was studied. (CTAB), ether, ethanol, CTAB, toluene and lysozyme were used to treat L. helveticus L7 with hexadecyltrimethylammonium bromide, respectively. The results showed that 20% ethanol was the best permeation treatment. When treated at room temperature for 30 min, the yield of permeable cells reached 468 渭 g / mL. The operating stability and storage stability of the crude linoleic acid isomerase solution prepared by ultrasonic cell fragmentation were studied. The results showed that the enzyme activity stability of the crude enzyme solution was poor. The enzyme activity decreased gradually at 4 鈩，

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